A healthy human depends on trillions of cells coordinating their activity for the benefit of the entire multicellular organism. When this regulatd cooperation between cells breaks down, disease occurs. Therefore, the study of the origin and maintenance of animal multicellularity will pave a stronger foundation for understanding the principles underlying a healthy human body. The origin and maintenance of multicellularity can be better understood through studying the primitive multicellularity exhibited by the closest livin relatives of animals-the choanoflagellate class of aquatic protists. The multicellular life stage o the choanoflagellate Salpingoeca rosetta was recently found to be induced by a sulfonolipid small molecule (RIF-1) that is produced by the choanoflagellate's bacterial prey Algoriphagus machipongonensis. However, it is still unclear what other multicellularity-inducing molecules are produced by dozens of other Bacteroidetes prey and also how the choanoflagellates respond to these molecules and RIF-1. Unveiling the range of multicellularity-inducing molecules and elucidating their choanoflagellate receptors will further the world's understanding of the foundations of animal multicellularity; therefore, the proposed research specifically aims at these objectives. First, multicellularity-inducing molecules produced by the marine Bacteroidetes bacteria Zobellia galactanivorans and Zobellia uliginosa will be isolated by extraction from bacterial cell pellets and activity- guided fractionation via high-performance liquid chromatography (HPLC). Newly discovered pure multicellularity-inducing molecules will be identified by standard spectroscopic methods. This work will demonstrate the breadth of molecules that induce choanoflagellate multicellularity. Subsequently, the biosynthetic pathways of the discovered multicellularity inducers will be hypothesized bioinformatically and confirmed by deleting the hypothesized biosynthesis genes from the Zobellia species and examining them for an inability to synthesize the target molecules. This uncovering of the genetic basis of multicellularity inducer synthesis will enable studies into the evolution of multicellularity-inducng genes across the bacterial kingdom-including bacteria in the human microbiota. Lastly, the choanoflagellate receptor proteins that bind to multicellularity-inducing molecules will be identified via synthesizing an affinity probe based on RIF-1 and using this probe to enrich for choanoflagellate proteins that bind multicellularity-inducing molecules. The proteins will be identified using mass spectrometry with comparison to the S. rosetta genome sequence. The identification of rosette inducer receptor proteins will enable studies into the evolutionary development and conservation of the proteins that drive multicellularity and respond to bacterial cues in animals. In total, this work will promote the world's understanding of the fundamental principles underlying animal multicellularity by uncovering both a range of new bacterially produced small molecule multicellularity cues and the choanoflagellate receptor proteins that recognize these cues.